Nucleic acids encoding lymphoid CD30 antigen

ABSTRACT

The description relates to the lymphoid surface antigen CD30 (Ki-1) occurring in Hodgkin&#39;s disease, the protein sequence and the associated nucleotide sequence therein, manufacture thereof by genetic engineering, methods for diagnosis and investigation of Hodgkin&#39;s disease and use of these nucleotide sequences for producing transgenic animals. The invention provides methods not based on monoclonal antibodies for investigation and diagnosis of anaplastic large-cell lymphomas.

This application is the U.S. National stage of PCT/DE92/00956, filedNov. 16, 1992.

The invention relates to the lymphoid surface antigen CD30 occurring inHodgkin's disease, its protein sequence and associated nucleotidesequence, manufacture thereof by genetic engineering, means fordiagnosis and investigation of Hodgkin's disease and use of thenucleotide sequences, more particularly for producing transgenicanimals.

The pathogenesis of Hodgkin's disease (HD) is as yet poorly understood,although in Central Europe it is numbered among the frequent humanlymphomas. After the surface antigen CD30 had been described,identification of the tumour cells of Hodgkin's disease, i.e. theHodgkin (H) and the Reed-Sternberg (RS) cells, became much easier orpossible for the first time (Schwab et al (1982), Nature 299, 65-67;Stein et al (1982) Int. J. Cancer 30, 445-449; McMichael, publisher(1987): Leucocyte Typing III, Oxford University Press, Oxford). Theappearance of the CD30 surface antigen is associated with activation ofthe lymphocytes, as shown by research on in vitro stimulated or virally(by HTLV-I, HTLV-II, EBV) transformed T and B cells (Stein et al, (1985)Blood, 66, 848-858; Andreesen et al. (1984) Blood, 63, 1299-1302). It isassumed that Hodgkin and Reed-Sternberg cells are malignant transformedT or B cells and that the histological variants in Hodgkin's disease aredue to secretion of different cytokines (Stein et al. (1989) RecentResults Cancer Res.117, 15-26). The CD30 antigen therefore seems to beimportant as regards the intracellular signal system and the genesis oftumours.

Histological investigation of tumour tissues has also shown that theCD30 antigen is suitable for identification of large-cell anaplasticneoplasms, which are therefore also called CD30 or Ki-1-positiveanaplastic large-cell lymphomas (ALCL; in English, Ki-1⁺ anaplasticlarge cell lymphomas). These tumours, which occur particularly inchildren and young people, constitute a separate species of lymphomasand are called true histiocytic lymphomas or malignant histiocytosis.The simultaneous occurrence of re-arranged Ig and T-cell receptor genesin most CD30 positive ALCLs shows that these tumour cells are lymphoid.Accordingly, CD30-positive ALCLs are not only a phenotypic but also agenotypic unit. CD30-positive ALCLs are classified as primary andsecondary; they originate mainly from T-cells and neutral cells, or to alesser extent from B-cells. Morphologically, ALCLs are classified as a)common; b) Hodgkin-related; c) giant cell-rich and d) lymphohistiocytic.

Hitherto, by definition, the CD30 antigen has been detectable only bymonoclonal antibodies, more particularly the monoclonal antibodies Ki-1and Ber-H2 (Schwab et al. (1982) Nature 299, 65-67; Schwarting et al(1989) Blood 74, 1678-1689). One disadvantage, however, is that theyonly recognise a respective epitope on the antigen; they are alsounsuitable for detecting and investigating the associated nucleic acids.A diagnosis based only on individual epitopes is also sensitive toartifacts and is not comprehensive. Finally, the recognisability of anisotope depends on how the sample is fixed and prepared; also theepitopes can alter with the state of the cell. For example an epitopecan be blocked by glycosylation of neighbouring N- or O-glycosylationsites, and also phosphorylation, more particularly tyrosinephosphorylation, etc., bring about conformation changes which cause anepitope to disappear, particularly if it is made up of a number ofpeptide-chain portions. Such changes in cell state occur particularlyoften during the progress of the tumour.

Hitherto also it has been impossible to make a detailed investigation ofthe properties of the CD30 antigen as a receptor or binding protein withregard to other growth or hormonal factors--partly because of the smallquantities available and also because the antigen is a membrane proteinwith functionally different cytosolic, transmembrane and extracellularprotein portions, hereinafter also called domains.

The object of the invention therefore is to provide means, not based onmonoclonal antibodies, for investigation, diagnosis and treatment ofHodgkin's disease and anaplastic large-cell lymphomas. More particularlythe object is to provide methods of investigating the protein functions,the development of tumours, and means for treating these diseases.

This problem is solved by an isolated nucleic acid--a DNA or RNA--whichcan be hybridised with the nucleotide sequence for the human lymphocyteactivation antigen CD30 shown in FIG. 2 SEQ ID NO:1. More particularlythe problem is solved by a DNA or RNA containing a sequence of thenucleotide sequence shown in FIG. 2.

The nucleic acid according to the invention can have modificationsdetectable by laboratory analysis, e.g. radioactive atoms, fluorescentstain groups (Hawkins & Sulston (1990) Technique, Dec. 307) and/orintroduced epitopes for light-based systems for detection of nucleicacids. In a preferred embodiment, the nucleic acids according to theinvention can be bonded to a macroscopic carrier, such as beads, moreparticularly glass or Sepharose bodies, membrane filters, nitrocellulosefilters or nylon filters, in order inter alia more easily to isolate orconcentrate CD30-RNA by hybridisation.

The invention also relates to cloning vectors containing anaforementioned DNA sequence. Particularly suitable cloning vectorsinclude those whereby the nucleic acids according to the invention canbe amplified, e.g. pBR322, pUR101, M13mp10, M13mp11, M13mp18, M13mp19,pGEM1, etc., or vectors for protein expression of the cloned DNAsequences in the host cells, e.g. lambda gt11, pATH, etc., or vectorswhich temporarily transform prokaryotic or eucaryotic host cells, e.g.PCDM8 or derivatives or pRC/CMV, etc., or vectors which transformeucaryotic host cells in stable manner and integrate into the hostgenome, e.g. pXT1, etc., or vectors suitable for integration in thegenome of germ cells and thus for producing transgenic animals, e.g.pSV2Neo, pRSVNeo, pMC1Neo, etc.,

The teaching according to the invention also covers host cells whichexpress CD30 antigen SEQ ID NO:2, a fragment of the CD30 antigen, ahybrid protein with CD30 antigen or mutations thereof, more particularlyeucaryotic host cells which incorporate CD30 antigen in the outer cellmembrane or provide it with post-translational modifications, e.g.glycosylate the CD30 antigen in natural manner or provide it withcomplex sugar molecules, e.g. the COS cells described in Example 3 orL929 cells, RK13 cells, WOP cells or R9ab cells.

In an embodiment of the invention, the aforementioned CD30 moleculesproduced by recombinant DNA methods are preferably immunogens forproducing polyclonal and monoclonal antibodies against the lymphoid CD30surface antigen. For example a hybrid protein with β-galactosidase andCD30 antigen fragments can be isolated from lambda-gt-11 clonesdescribed hereinafter and used as an immunogen. The aforementionedmethod can also be used to express CD30 antigen in eucaryotic cells anduse it for immunisation, after purification or as a protein in themembrane together with the over-expressing cell. Also, CD30 peptidescorresponding to the protein sequence shown in FIG. 2 can be synthesisedby chemical means, optionally coupled by glutaraldehyde or anothercoupling reagent to a suitable carrier, e.g. thyreoglobulin or albumin,and injected as an immunogen together with Freund adjuvant into asuitable animal, such as a mouse, rabbit, goat or sheep. CD30 (hybrid)molecules produced by biological or chemical synthesis are also suitablefor use in the so-called "sandwich immuno-absorption" test.

According to another feature of the invention, pharmacologicallyrelevant parts of CD30 are also made manifest by a knowledge of thenucleotide and protein sequence. Of particular pharmacologicalimportance are the extracellular regions of the CD30 molecule, sinceligand molecules to CD30 administered for therapeutic purposes naturallybecome bound first to the extracellular part of the membrane protein.The extracellular regions of CD30, as shown by comparative studies, thehydropathy curve and in vitro transcription and translationinvestigations, are coded mainly by the cDNA sequence between position277 and position 1359, i.e. the sequence between ²⁷⁷ -TTCCCACAGGATCGACCC. . . (SEQ ID NO:3) and . . . CTCCCACGGGGAAG₁₃₅₉ (SEQ ID NO:4). It istherefore an obvious idea to use these regions of the CD30 molecule orsub-sections thereof in particular for development of therapeuticagents. In particular, fusion proteins with original extracellular CD30portions are particularly suitable for isolating or concentratingnatural or synthetic immunotoxins against Hodgkin and Reed-Sternbergcells. Also, oligopeptides or recombinant peptides prepared on the basisof extracellular amino acid sequences of CD30 can be used as immunogensfor producing polyclonal antibodies against extracellular CD30. Also,synthetic ligands to these protein sequences can be produced ordeveloped. These ligands or antibodies can be coupled with a toxin, suchas saponin or ricin A, so as to obtain therapeutically usefulimmunotoxins for treating these lymphomas. The application thereforealso relates to use of the extracellular CD30 sequences for manufacture,search or isolation of binding partners, i.e. natural, already existingin vivo, or artificial, produced in vitro or induced in vivo,particularly ligands for producing immunotoxins. Special importanceattaches to polyclonal antibodies against extracellular CD30 sequences,so that now for the first time polyclonal immunotoxins are availableagainst Hodgkin's disease and anaplastic large-cell lymphomas. Theextracellular CD30 peptide sequences can be produced by chemicalsynthesis, more particularly solid-phase peptide synthesis, or asrecombinant fusion protein in prokaryotic and eucaryotic cells.Expression in yeasts and eucaryotic cells is preferred, particularly ininsect cells or in tissue culture with fibroblasts, preferably humanfibroblasts, since in that case the extracellular protein cells areglycosylated and modified partly in natural manner.

The invention also relates to use of theintracellular--cytosolic--protein sequences of CD30 which contribute tointracellular signal transduction. Clearly these sequences code for aportion of use for interaction with other proteins involved inadditional signal transduction. The cytosolic part of the CD30participating in intracellular signal transduction, as shown bycomparative studies, the hydropathy curve and in vitro transcription andtranslation research, is mainly coded by the nucleotide sequence (seeFIG. 2) between position 1444 and 2007, i.e. the sequence between ¹⁴⁴⁴-CACCGGAGGGCCTGCA (SEQ ID NO:5) . . . and . . . CTGCCTCTGGAAAG-₂₀₀₇ (SEQID NO:6). Another important aspect of this feature of the invention isthe synthesis of recombinant CD30 protein (portions) with cytosolicactivity, and development of ligands and bonding partners whichinfluence the cytosolic activity of CD30.

The teaching according to the invention also covers use of theaforementioned nucleotide sequences for producing transgenic animals,more particularly rodents, with changed CD30 antigen expression, i.e.mutated, more particularly truncated CD30, over or under-expression ofCD30, expression of CD30 in cells and tissues other than lymphocytes,complete absence of an intact structural gene for CD30, constitutive orinducible expression of CD30, or expression of CD30 at a predeterminedsite during differentiation of the cells. Such transgenic animals areexcellent models for scientific research on lymphomas, development ofclinical tests, or research into blocking and inductor molecules for theextracellular receptor part of the CD30 antigen.

Other preferred embodiments of the invention are described in thesubsidiary and sub-claims.

The means and methods provided by the teaching according to theinvention can now be used for detection of CD30 antigen-coding DNAs andRNAs by hybridisation or for localisation of the gene on the chromosomeor chromosomes. It is therefore possible to investigate whethertranscription of the CD30 gene has occurred in the cells in the sample,and also to investigate the regulation mechanisms for expression of theCD30 antigen.

In addition to these more scientific applications, the novelhybridisation methods--the PCR (polymerase chain) reaction, in-situhybridisation on portions of tissue and chromosomes--provide anadditional, very sensitive diagnosis. In particular, this diagnosis isnot sensitive to post-translational modifications and processings.

Investigations on chromosomal localisation of the CD30 structural genehave at least shown that the gene lies in the chromosomal band 1p36,i.e. at a fragile place in the chromosome (see LeBeau (1986) Blood 67,849-858) where chromosomal abnormalities including virus insertions(EBV, HTLVI and II, HV6 etc.) have already been detected in Hodgkin'sdisease and non-Hodgkin lymphomas and other neoplasias such asneuroblastomas and malignant melanomas. More particularly, in non-Hodgkin lymphomas, changes (duplication, translocations, deletions,etc.) occur in this chromosomal region and, together with expression ofthe CD30 antigen, are associated with particularly rapid growth oftumour. The nucleotide sequences provided by the invention are thereforeexcellent means for diagnosing whether these chromosomal changes haveoccurred in the patient and whether Hodgkin's disease or relatedlarge-cell anaplastic lyphomas are likely to occur.

The nucleotide sequences provided by the invention are also suitable forprenatal diagnosis, particularly when such diseases have alreadyoccurred in the family.

Other advantages, embodiments and features of the invention will beclear from the following description in conjunction with the drawingsand diagrams, in which:

FIG. 1A) shows cloned CDNA portions of CD30 antigen mRNA in lambda gt11;

FIG. 1B) shows the regions (shaded rectangles) coding for the CD30antigen on the mRNA and the non-coding regions (lines) with thepolyadenylation sites (asterisks),

FIG. 1C) is a restriction map of the cDNA with various restrictionenzymes;

FIG. 1D) is a map of the CD30 antigen based on epitopes of themonoclonal antibodies Ki-1 and Ber-H2;

FIG. 2 parts A-H show the sequence of the non-transcribed strand of DNA,the coding strand of DNA and the CD30 protein sequence derivabletherefrom;

FIG. 3 shows research into CD30-mRNAs in various human cell lines;

FIG. 4 is a diagram of the hydrophobic and hydrophilic properties of theCD30 amino acid sequence after Kyte & Doolittle (1982) J. Mol. Biol.157,105-132;

FIG. 5 compares the CD30 sequence with sequences from the NGF receptorprotein family. (SEQ ID NOS. 7-11)

FIG. 6 shows expression of CD30 in COS cells.

immune precipitation of cells labelled with ¹²⁵ J:

track 1: marker proteins

track 2: BER-H2 precipitate of CD30-5 transfected COS cells

track 3: BER-H2 precipitate of HUT-102 cells

track 4: BER-H2 precipitate of mock (PCDM8) transfected COS cells.

Cloning and Research into CD30-cDNA

A lambda-gt11-cDNA gene library of HUT-102 cells (Poiesz et al (1980)Proc. Natl. Acad. Sci. USA, 77, 7415-7419) comprising about 200 000clones was tested with monoclonal antibodies for hybrid proteins withCD30 antigen.

cDNA synthesis based on poly(A)⁺ -RNA from HUT-102 cells was carried outafter Guebler & Hoffmann (1983), Gene 25, 263-269. By way of variation,the cDNA was size-selected in a Sepharose-CL4B column (Eschenfeldt &Berger (1987) Methods in Enzymol.152, 335-337). Immunoscreening wasperformed with a mixture of the monoclonal antibody Ki-1 and Ber-H2after Huynh et al (1985) in "DNA Cloning", A Practical Approach, Vol. 1,49-78 (Published by D. M. Glover, IRL Press, Oxford, Washington D.C.).

A clone with a 909 bp CDNA produced a hybrid peptide which wasrecognised by the monoclonal antibody Ber-H2.

In order to obtain a clone containing the entire sequence for CD30, apCDM8 gene library from HUT-102 cells was prepared (Seed & Aruffo (1987)Proc. Natl. Acad. Sci. USA 84, 3365-3369; Aruffo & Seed (1987) Proc.Natl. Acad. Sci. USA. 84, 8573-8577). The cDNA was synthesised using acDNA synthesis kit produced by the company Invitrogen, then providedwith BstXI linkers and finally size-fractionated on a 1.5% (w/v)Nu-Sieve gel made by the company FMC. cDNA with more than 1 kbp wasisolated from the gel and ligated with the vector pCDM8. Transformationwas brought about in E.coli- MC1061/p3 by electroporation after Dover etal (1988) Nucl. Acids. Res. 16, 6127-6145.

The colonies were hybridised by using the CD30-1-cDNA sample obtained byimmunoscreening the lambda-gt11 library (T. Maniatis, E. F. Fritsch andJ. Sambrook (1989) Molecular Cloning: A laboratory manual; Cold SpringHarbour Press, Cold Spring Harbour, New York) . This approach yielded 5clones overlapping in the cDNA sequences (FIG. 1) with 1492 bp (CD30-2),1905 bp (CD30-3 and CD30-4), 2342 bp (CD30-5) and 2242 (CD30- 6). DNAwas sequenced by using the Sequenase Sequencing Kit by the company USBiochemicals and synthetic oligonucleotide primers (Sanger et al (1977)Proc. Natl. Acad. Sci. USA, 74, 5463-5467).

In order to clone the 3'-poly(A) end also, a PCR reaction according tothe RACE (rapid amplification of cDNA ends) protocol was brought abouton reverse-transcribed poly(A)RNA from HUT-102 cells (Frohman et al(1988), Proc. Natl. Acad. Sci. USA, 85, 8998-9002). Amplification wasbrought about in 35 cycles comprising: 1 minute denaturing followed by45 seconds addition of the primer at 54° C. and 1.5 minutes elongationat 72° C., with internal primers and oligo(dT) primers comprising a Sal1site. The reaction was performed in 50 mM Tris HCl, pH 8.15, 6 mM MgCl₂,40 mM KCl. This process yielded clone CD30-7 with a 333 bp cDNA.

The DNA of clone CD30-5 in vector pGEM1 was deposited, number DSM 6833,at the DSM (=German Collection of Microorganisms and Cell Cultures) inaccordance with the Budapest Agreement.

The sequence of the CD30-cDNA and the protein sequence derivabletherefrom are shown in FIG. 2. The signal peptide and the transmembranedomains are twice underlined. Polyadenylation signals are underlinedonce. The polyadenylation sites are marked by arrows. The homologoussub-units in the extracellular domains are enclosed in a frame,potential N-glycosylation sites are marked by asterisks, and potentialphosphorylation sites are marked by the following abbreviations: TYR(tyrosine kinase), PKC (protein kinase C), CK2 (casein kinase II) andAMP (cAMP./cGMP-dependant kinase).

The complete nucleotide sequence of CD30-cDNA contains 3630 base pairs,the G/C content being 62%. The open reading frame goes from nucleotide223 to nucleotide 2007--the termination codon. The ATG codon forinitiation of translation is surrounded by typical start sequences(Kozak (1986) Cell 44, 283-292). The open reading frame consists of two77% homologous approx. 360 bp domains (367-747 and 995-1272). The openreading frame is preceded by 230 base pairs of non-translated leadersequence, followed by 1613 base pairs of untranslated sequence with ashort palindromic sequence between nucleotide 2867 and 2888. Theunusually long 5' leader sequence is unnecessary for expression, since acDNA without this sequence, e.g. from clone CD30-5 (see Example 3), canbe expressed in COS cells.

The untranslated 3' region contains two sites for polyadenylation(marked by an arrow in FIG. 2), preceded by the unusual poly(A) signalsequences TGTAAA and AATAAT. (Birnstiel et al (1985) Cell 41, 349-359);Bardwell et al (1985) Cell 65, 125-133; Sheets et al (1990) Nucl. Acids.Res.18, 5799-5805).

Use of CD30 Nucleotide Sequences for Investigation of Cells, Tissues andChromosomes

EXAMPLE 1

Northern blot analyses. The RNA from the cells and tissues was isolatedby the guanidinium isothiocyanate-caesium chloride method of Chirgwin etal (1979) Biochemistry, 18, 5294-5299. The RNA was purified by anoligo(dT) cellulose column after Swan et al (1972) Proc. Natl. Acad.Sci. USA 69, 1408-1412. The resulting poly(A)⁺ RNA was then separated bygel electrophoresis on a 1.2% (w/v) agarose gel with 2.2 mol/lformaldehyde--2 μg poly(A)⁺ RNA per track--transferred to nitrocellulosemembranes and then hybridised against ³² P-dCTP labelled CD30 DNAinserts. The DNA was radioactively labelled by using a "DNA labellingkit" by Boehringer/Mannheim. Hybridisation was brought about by themethod of Thomas (1977) Proc. Natl. Acad. Sci. USA, 77, 5201-5205.

FIGS. 3A and B show this kind of Northern blot analysis of RNA from thehuman cell lines HUT-102, SU-DHL-1 (Morgan et al (1989) Blood 73,2155-2164), L-428 (Schaadt et al (1980) Int. J. Cancer 26, 723-731,L-591 (Diehl et al (1982) Cancer Treat. Rep.66, 615-632), L-540 (Diehlet al (1981) J. Cancer Res. Clin. Oncol.101, 111-124), Co (Jones et al.(1985) Hematol. Oncol.3, 133-145), Ho (Kamesaki et al (1986) Blood 68,285-292), U-937 (Sundstrom & Nilsson (1976) Int. J. Cancer 17, 565-577),MDA-MB-231 (Cailleau et al. (1974) J. Nat. Cancer Inst. 53, 661-674) andHPB-ALL (Boylston & Cosford (1985) Eur. J. Immunol. 15, 738-742) and,for comparison, of PBL cells (peripheral blood lymphocytes)). Theautoradiography lasted four days. The hybridisation sample was theCD30-5 insert with the coding region (FIG. 3A) and with the CD30-6/Bgl1-Xba1 fragment of the non-translated 3' end (3B).

Two RNAs were detected in the cell lines under investigation, a largerRNA with about 3800 nucleotides and a smaller with about 2600nucleotides. The RNAs probably originate from different splicing duringpolyadenylation.

The amount of CD30-coding RNA was greatest in the cell line SU-DHL-1 andin the cell line L-591 originating from a Hodgkin's lymphoma. It wasfollowed by the cell lines L-540, L-428, Ho, Co originating fromHodgkin's lymphomas and finally by the cell line HUT-102 transformed byHTLV-1. Corresponding to the appearance of the CD30 antigen, no RNAtranscripts were present in the peripheral blood lymphocytes or in thecell lines HPB-ALL, MDA-MB-231 or U-937. This shows the high specificityand sensitivity of the hybridisation sample.

EXAMPLE 2

Mapping of chromosomes. Isolated peripheral blood lymphocytes stimulatedwith PHA and from 2 healthy donors were blocked in culture by colchicineduring cell division in the metaphase after Stollmann et al (1985), Br.J. Haematol. 60, 183-196 and the chromosomes were prepared, followed byin situ hybridisation after Fonatsch et al (1987), Cytogenet. CellGenet. 45, 109-112 with the CD30-5/Smal-cDNA fragment containing thesequence between the Smal site at position 218 and the Smal site atposition 1732. The DNA was radioactively labelled by nick-translationwith ³ H-dCTP and ³ H-dTTP. The specific activity of the sample was1.2×10⁷ dpm/μg DNA. Hybridization is performed in 2X SSC, 10% dextransulfate and 50% formamide at 37° C. Washes are performed in 2X SSC, 50%formamide at 40° C., then in 2X SSC at room temperature. 99 metaphaseswere counted.

This research showed that the CD30 gene lies on the chromosome band1p36. 21.6% of 162 silver grains lay specifically in this region. Thechi-squared values are highly significant (P<0.001).

EXAMPLE 3

Expression of CD30 antigens in COS cells. To this end, COS cells weretransfected with CD30-5-cDNA, after which the CD30 antigen wasimmune-precipitated from these cells. The expression vector used was thepCDM8 vector, in which the CD30-cDNA piece had been cloned. Transfectionwas brought about on COS-1 cells in cell culture by the DEAE dextranmethod (Seed & Aruffo (1987) Proc. Natl. Acad. Sci. USA, 84 3365-3369).48 hours after transfection the cells were isolated, radioactivelylabelled with IODO beads (Messrs Pierce) and solubilised, and theproteins were immune-precipitated and separated by gel electrophoresis.

As a control, surface proteins from HUT-102 cells were radioactivelylabelled with IODO beads (produced by the company Pierce) and isolatedby means of agarose beads coated with anti-mouse Ig (Schwarting et al(1989) Blood 74, 1678-1689).

FIG. 6 shows electrophoretic SDS gel analysis of CD30immune-precipitated with monoclonal antibody Ber-H2. Track 1: control.Track 2: CD30-5-cDNA transfected COS cells. Track 3: From ¹²⁵ -labelledHUT-102 cells. Track 4: control--COS cells transfected with vectorwithout insert. In all cases a single protein band with a relativemolecular weight of 120 000 was found.

Immune-cytological APAAP (alkaline phosphatase anti-alkalinephosphatase) staining (Cordell et al (1984) J. Histochem. Cytochem. 32219-229) was also carried out. This was a method of mapping the epitopesof monoclonal antibodies Ki-1 and Ber-H2 by comparison to show whichmonoclonal antibodies reacted with which hybrid proteins or truncatedexpressed protein, and was also a method of mapping the amino acidsequence derived from the cDNA map. The data from lambda gt11immuno-screening and APAAP immune staining can be combined in order tolocalise the Ki-1 epitope in the extracellular domain (see FIG. 1D)between the amino terminus and the amino acid 93, and the Ber-H2 epitopeis localised between the amino acids 112 and 416.

EXAMPLE 4

Expression of the CD30 antigen in rabbit cell lines RK13 and R9ab forimmunising rabbits so as to produce polyclonal sera against CD30. Tothis end the cell lines were transfected with the vector pCDM8-CD30-5 orpRC/CMV-CD30-5 DEAE dextran. After transfection the cells were harvestedand used to immunise rabbits in accordance with a suitable protocol.

EXAMPLE 5

Expression of the CD30 antigen in mouse fibroblast cell lines L929 andWOP for producing additional monoclonal antibodies with rats and miceagainst CD30. To this end the mouse fibroblasts were transfected withthe vector pCDM8-CD30-5 or pRC/CMV-CD30-5 by the DEAE dextran method orby spheroblast fusion (Sandri-Goldrin et al Mol. Cell. Biol. 1 743-752).After transfection, mice or rats were immunised with the cells inaccordance with a suitable protocol. In order to produce the hybridomasthe spleens of the animals were prepared and the resulting lymphocyteswere fused with a suitable mouse myeloma cell line (G. Kohler, C.Milstein, Nature (1975), 256, 495-497).

EXAMPLE 6

Use of CD30 antigen obtained by a recombinant method in RIAs, ELISAs,EIAs or similar immunodetection systems for measuring the soluble CD30antigen occurring in sera during certain diseases. The CD30 for thispurpose can be obtained by expression in eucaryotes or procaryotes,using the stated nucleic acid sequence, slightly modified if necessary.After affinity-chromatographic purification and determination of theCD30 content, these preparations can be used for standardisation of CD30immunoassays.

The CD30 Peptide Sequence and the Resulting Possible Applications of theInvention

A comparison involving the derived amino acid sequence for the CD30antigen shows that the CD30 polypeptide is equivalent to othertransmembrane growth factor receptors. More particularly the first and,to a lesser extent, the second cysteine-rich sub-unit in theextracellular domain is equivalent to the extracellular domains of otherknown receptors (see FIG. 5). The main similarities are with the humannerve growth factor receptor protein family, e.g. NGFR, TNFR-1, TNFR-II(Schall et al (1990) Cell 61, 361-370; Heller et al (1990) Proc. Natl.Acad. Sci. USA 87, 6151-6155; Loetscher et al (1990) Cell 61, 351-359;Kohno et al (1990) Proc. Natl. Acad. Sci. USA 87, 8331-8335; Engelmannet al (1990), J. Biol. Chem. 265, 1531-1536; Smith et al (1990) Science248, 1019-1023) and the human nerve growth factor receptor (NGFR) of lowaffinity (Johnson et al (1986) Cell 47, 545-554).

The extracellular domain of CD30 can be divided into six cysteine-richprotein motives of 40 amino acids (see FIG. 4) which, apart from theshortened motives 1B and 3B, each contain six cysteine radicals. As inthe case of other members of the NGFR protein family, the positions ofthe cysteines in these sub-domains are highly conserved. There are alsohomologies to the corresponding regions of the following receptors:human insulin receptor-related receptor; Shier & Watt (1989), J. Biol.Chem. 264, 14605-14608); Transforming growth factor-β1 binding protein;Kanzaki et al (1990), Cell 61, 1051-1061); Mouse interleukin-3 receptor)Gorman et al (1990) Proc. Natl. Acad. Sci. USA 87, 5459-5463); sevenlessprotein of the fruit fly (Michael et al (1990) Proc. Natl. Acad. Sci.USA 87, 5351-5353).

At the DNA level there are homologies to other growth factor receptors,particularly the human fibroblast growth factor receptor-4 (Partanen etal (1991) EMBO J. 10, 1347-1354).

The results of comparisons between sequences, therefore, show that theCD30 antigen also is very probably a receptor for a growth factor.Consequently the nucleotide and protein sequences of CD30 provided bythe invention can obviously be used for receptor studies, i.e. whichfactors can become bound to CD30, how--competitively orconstitutively--the bond can be influenced, and what effects the bondingof the ligands have on the cell.

More particularly, the nucleotide sequence made available can be used tocreate transgenic animals for investigation of the receptor and theassociated signal system.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 11    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 3627 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Homo sapiens    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 223..2010    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    ATACGGGAGAACTAAGGCTGAAACCTCGGAGGAACAACCACTTTTGAAGTGACTTCGCGG60    CGTGCGTTGGGTGCGGACTAGGTGGCCCCGGCGGGAGTGTGCTGGAGCCTGAAGTCCACG120    CGCGCGGCTGAGAACCGCCGGGACCGCACGTGGGCGCCGCGCGCTTCCCCCGCTTCCCAG180    GTGGGCGCCGGCCGCCAGGCCACCTCACGTCCGGCCCCGGGGATGCGCGTCCTC234    MetArgValLeu    CTCGCCGCGCTGGGACTGCTGTTCCTGGGGGCGCTACGAGCCTTCCCA282    LeuAlaAlaLeuGlyLeuLeuPheLeuGlyAlaLeuArgAlaPhePro    5101520    CAGGATCGACCCTTCGAGGACACCTGTCATGGAAACCCCAGCCACTAC330    GlnAspArgProPheGluAspThrCysHisGlyAsnProSerHisTyr    253035    TATGACAAGGCTGTCAGGAGGTGCTGTTACCGCTGCCCCATGGGGCTG378    TyrAspLysAlaValArgArgCysCysTyrArgCysProMetGlyLeu    404550    TTCCCGACACAGCAGTGCCCACAGAGGCCTACTGACTGCAGGAAGCAG426    PheProThrGlnGlnCysProGlnArgProThrAspCysArgLysGln    556065    TGTGAGCCTGACTACTACCTGGATGAGGCCGACCGCTGTACAGCCTGC474    CysGluProAspTyrTyrLeuAspGluAlaAspArgCysThrAlaCys    707580    GTGACTTGTTCTCGAGATGACCTCGTGGAGAAGACGCCGTGTGCATGG522    ValThrCysSerArgAspAspLeuValGluLysThrProCysAlaTrp    859095100    AACTCCTCCCGTGTCTGCGAATGTCGACCCGGCATGTTCTGTTCCACG570    AsnSerSerArgValCysGluCysArgProGlyMetPheCysSerThr    105110115    TCTGCCGTCAACTCCTGTGCCCGCTGCTTCTTCCATTCTGTCTGTCCG618    SerAlaValAsnSerCysAlaArgCysPhePheHisSerValCysPro    120125130    GCAGGGATGATTGTCAAGTTCCCAGGCACGGCGCAGAAGAACACGGTC666    AlaGlyMetIleValLysPheProGlyThrAlaGlnLysAsnThrVal    135140145    TGTGAGCCGGCTTCCCCAGGGGTCAGCCCTGCCTGTGCCAGCCCAGAG714    CysGluProAlaSerProGlyValSerProAlaCysAlaSerProGlu    150155160    AACTGCAAGGAACCCTCCAGTGGCACCATCCCCCAGGCCAAGCCCACC762    AsnCysLysGluProSerSerGlyThrIleProGlnAlaLysProThr    165170175180    CCGGTGTCCCCAGCAACCTCCAGTGCCAGCACCATGCCTGTAAGAGGG810    ProValSerProAlaThrSerSerAlaSerThrMetProValArgGly    185190195    GGCACCCGCCTCGCCCAGGAAGCTGCTTCTAAACTGACGAGGGCTCCC858    GlyThrArgLeuAlaGlnGluAlaAlaSerLysLeuThrArgAlaPro    200205210    GACTCTCCCTCCTCTGTGGGAAGGCCTAGTTCAGATCCAGGTCTGTCC906    AspSerProSerSerValGlyArgProSerSerAspProGlyLeuSer    215220225    CCAACACAGCCATGCCCAGAGGGGTCTGGTGATTGCAGAAAGCAGTGT954    ProThrGlnProCysProGluGlySerGlyAspCysArgLysGlnCys    230235240    GAGCCCGACTACTACCTGGACGAGGCCGGCCGCTGCACAGCCTGCGTG1002    GluProAspTyrTyrLeuAspGluAlaGlyArgCysThrAlaCysVal    245250255260    AGCTGTTCTCGAGATGACCTTGTGGAGAAGACGCCATGTGCATGGAAC1050    SerCysSerArgAspAspLeuValGluLysThrProCysAlaTrpAsn    265270275    TCCTCCCGCACCTGCGAATGTCGACCTGGCATGATCTGTGCCACATCA1098    SerSerArgThrCysGluCysArgProGlyMetIleCysAlaThrSer    280285290    GCCACCAACTCCTGTGCCCGCTGTGTCCCCTACCCAATCTGTGCAGCA1146    AlaThrAsnSerCysAlaArgCysValProTyrProIleCysAlaAla    295300305    GAGACGGTCACCAAGCCCCAGGATATGGCTGAGAAGGACACCACCTTT1194    GluThrValThrLysProGlnAspMetAlaGluLysAspThrThrPhe    310315320    GAGGCGCCACCCCTGGGGACCCAGCCGGACTGCAACCCCACCCCAGAG1242    GluAlaProProLeuGlyThrGlnProAspCysAsnProThrProGlu    325330335340    AATGGCGAGGCGCCTGCCAGCACCAGCCCCACTCAGAGCTTGCTGGTG1290    AsnGlyGluAlaProAlaSerThrSerProThrGlnSerLeuLeuVal    345350355    GACTCCCAGGCCAGTAAGACGCTGCCCATCCCAACCAGCGCTCCCGTC1338    AspSerGlnAlaSerLysThrLeuProIleProThrSerAlaProVal    360365370    GCTCTCTCCTCCACGGGGAAGCCCGTTCTGGATGCAGGGCCAGTGCTC1386    AlaLeuSerSerThrGlyLysProValLeuAspAlaGlyProValLeu    375380385    TTCTGGGTGATCCTGGTGTTGGTTGTGGTGGTCGGCTCCAGCGCCTTC1434    PheTrpValIleLeuValLeuValValValValGlySerSerAlaPhe    390395400    CTCCTGTGCCACCGGAGGGCCTGCAGGAAGCGAATTCGGCAGAAGCTC1482    LeuLeuCysHisArgArgAlaCysArgLysArgIleArgGlnLysLeu    405410415420    CACCTGTGCTACCCGGTCCAGACCTCCCAGCCCAAGCTAGAGCTTGTG1530    HisLeuCysTyrProValGlnThrSerGlnProLysLeuGluLeuVal    425430435    GATTCCAGACCCAGGAGGAGCTCAACGCAGCTGAGGAGTGGTGCGTCG1578    AspSerArgProArgArgSerSerThrGlnLeuArgSerGlyAlaSer    440445450    GTGACAGAACCCGTCGCGGAAGAGCGAGGGTTAATGAGCCAGCCACTG1626    ValThrGluProValAlaGluGluArgGlyLeuMetSerGlnProLeu    455460465    ATGGAGACCTGCCACAGCGTGGGGGCAGCCTACCTGGAGAGCCTGCCG1674    MetGluThrCysHisSerValGlyAlaAlaTyrLeuGluSerLeuPro    470475480    CTGCAGGATGCCAGCCCGGCCGGGGGCCCCTCGTCCCCCAGGGACCTT1722    LeuGlnAspAlaSerProAlaGlyGlyProSerSerProArgAspLeu    485490495500    CCTGAGCCCCGGGTGTCCACGGAGCACACCAATAACAAGATTGAGAAA1770    ProGluProArgValSerThrGluHisThrAsnAsnLysIleGluLys    505510515    ATCTACATCATGAAGGCTGACACCGTGATCGTGGGGACCGTGAAGGCT1818    IleTyrIleMetLysAlaAspThrValIleValGlyThrValLysAla    520525530    GAGCTGCCGGAGGGCCGGGGCCTGGCGGGGCCAGCAGAGCCCGAGTTG1866    GluLeuProGluGlyArgGlyLeuAlaGlyProAlaGluProGluLeu    535540545    GAGGAGGAGCTGGAGGCGGACCATACCCCCCACTACCCCGAGCAGGAG1914    GluGluGluLeuGluAlaAspHisThrProHisTyrProGluGlnGlu    550555560    ACAGAACCGCCTCTGGGCAGCTGCAGCGATGTCATGCTCTCAGTGGAA1962    ThrGluProProLeuGlySerCysSerAspValMetLeuSerValGlu    565570575580    GAGGAAGGGAAAGAAGACCCCTTGCCCACAGCTGCCTCTGGAAAGTGA2010    GluGluGlyLysGluAspProLeuProThrAlaAlaSerGlyLys*    585590595    GGCCTGGGCTGGGCTGGGGCTAGGAGGGCAGCAGGGTGGCCTCTGGGAGGCCAGGATGGC2070    ACTGTTGGCACCGAGGTTGGGGGCAGAGGCCCATCTGGCCTGAACTGAGGCTCCAGCATC2130    TAGTGGTGGACCGGCCGGTCACTGCAGGGGTCTGGTGGTCTCTGCTTGCATCCCCAACTT2190    AGCTGTCCCCTGACCCAGAGCCTAGGGGATCCGGGGCTTGTACAGAAGAGACAGTCCAAG2250    GGGACTGGATCCCAGCAGTGATGTTGGTTGAGGCAGCAAACAGATGGCAGGATGGGCACT2310    GCCGAGAACAGCATTGGTCCCAGAGCCCTGGGCATCAGACCTTAACCACCAGGCCCACAG2370    CCCAGCGAGGGAGAGGTCGTGAGGCCAGCTCCCGGGGCCCCTGTAACCCTACTCTCCTCT2430    CTCCCTGGACCTCAGAGGTGACACCCATTGGGCCCTTCCGGCATGCCCCCAGTTACTGTA2490    AATGTGGCCCCCAGTGGGCATGGAGCCAGTGCCTGTGGTTGTTTCTCCAGAGTCAAAAGG2550    GAAGTCGAGGGATGGGGCGTCGTCAGCTGGCACTGTCTCTGCTGCAGCGGCCACACTGTA2610    CTCTGCACTGGTGTGAGGGCCCCTGCCTGGACTGTGGGACCCTCCTGGTGCTGCCCACCT2670    TCCCTGTCCTGTAGCCCCCTCGGTGGGCCCAGGGCCTAGGGGCCCAGGATCAAGTCACTC2730    ATCTCAGAATGTCCCCACCAATCCCCGCCACAGCAGGCGCCTCGGGTCCCAGATGTCTGC2790    AGCCCTCAGCAGCTGCAGACCGCCCCTCACCAACCCAGAGAACCTGCTTTACTTTGCCCA2850    GGGACTTCCTCCCCATGTGAACATGGGGAACTTCGGGCCCTGCCTGGAGTCCTTGACCGC2910    TCTCTGTGGGCCCCACCCACTCTGTCCTGGGAAATGAAGAAGCATCTTCCTTAGGTCTGC2970    CCTGCTTGCAAATCCACTAGCACCGACCCCACCACCTGGTTCCGGCTCTGCACGCTTTGG3030    GGTGTGGATGTCGAGAGGCACCACGGCCTCACCCAGGCATCTGCTTTACTCTGGACCATA3090    GGAAACAAGACCGTTTGGAGGTTTCATCAGGATTTTGGGTTTTTCACATTTCACGCTAAG3150    GAGTAGTGGCCCTGACTTCCGGTCGGCTGGCCAGCTGACTCCCTAGGGCCTTCAGACGTG3210    TATGCAAATGAGTGATGGATAAGGATGAGTCTTGGAGTTGCGGGCAGCCTGGAGACTCGT3270    GGACTTACCGCCTGGAGGCAGGCCCGGGAAGGCTGCTGTTTACTCATCGGGCAGCCACGT3330    GCTCTCTGGAGGAAGTGATAGTTTCTGAAACCGCTCAGATGTTTTGGGGAAAGTTGGAGA3390    AGCCGTGGCCTTGCGAGAGGTGGTTACACCAGAACCTGGACATTGGCCAGAAGAAGCTTA3450    AGTGGGCAGACACTGTTTGCCCAGTGTTTGTGCAAGGATGGAGTGGGTGTCTCTGCATCA3510    CCCACAGCCGCAGCTGTAAGGCACGCTGGAAGGCACACGCCTGCCAGGCAGGGCAGTCTG3570    GCGCCCATGATGGGAGGGATTGACATGTTTCAACAAAATAATGCACTTCCTTAAAAA3627    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 595 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetArgValLeuLeuAlaAlaLeuGlyLeuLeuPheLeuGlyAlaLeu    151015    ArgAlaPheProGlnAspArgProPheGluAspThrCysHisGlyAsn    202530    ProSerHisTyrTyrAspLysAlaValArgArgCysCysTyrArgCys    354045    ProMetGlyLeuPheProThrGlnGlnCysProGlnArgProThrAsp    505560    CysArgLysGlnCysGluProAspTyrTyrLeuAspGluAlaAspArg    65707580    CysThrAlaCysValThrCysSerArgAspAspLeuValGluLysThr    859095    ProCysAlaTrpAsnSerSerArgValCysGluCysArgProGlyMet    100105110    PheCysSerThrSerAlaValAsnSerCysAlaArgCysPhePheHis    115120125    SerValCysProAlaGlyMetIleValLysPheProGlyThrAlaGln    130135140    LysAsnThrValCysGluProAlaSerProGlyValSerProAlaCys    145150155160    AlaSerProGluAsnCysLysGluProSerSerGlyThrIleProGln    165170175    AlaLysProThrProValSerProAlaThrSerSerAlaSerThrMet    180185190    ProValArgGlyGlyThrArgLeuAlaGlnGluAlaAlaSerLysLeu    195200205    ThrArgAlaProAspSerProSerSerValGlyArgProSerSerAsp    210215220    ProGlyLeuSerProThrGlnProCysProGluGlySerGlyAspCys    225230235240    ArgLysGlnCysGluProAspTyrTyrLeuAspGluAlaGlyArgCys    245250255    ThrAlaCysValSerCysSerArgAspAspLeuValGluLysThrPro    260265270    CysAlaTrpAsnSerSerArgThrCysGluCysArgProGlyMetIle    275280285    CysAlaThrSerAlaThrAsnSerCysAlaArgCysValProTyrPro    290295300    IleCysAlaAlaGluThrValThrLysProGlnAspMetAlaGluLys    305310315320    AspThrThrPheGluAlaProProLeuGlyThrGlnProAspCysAsn    325330335    ProThrProGluAsnGlyGluAlaProAlaSerThrSerProThrGln    340345350    SerLeuLeuValAspSerGlnAlaSerLysThrLeuProIleProThr    355360365    SerAlaProValAlaLeuSerSerThrGlyLysProValLeuAspAla    370375380    GlyProValLeuPheTrpValIleLeuValLeuValValValValGly    385390395400    SerSerAlaPheLeuLeuCysHisArgArgAlaCysArgLysArgIle    405410415    ArgGlnLysLeuHisLeuCysTyrProValGlnThrSerGlnProLys    420425430    LeuGluLeuValAspSerArgProArgArgSerSerThrGlnLeuArg    435440445    SerGlyAlaSerValThrGluProValAlaGluGluArgGlyLeuMet    450455460    SerGlnProLeuMetGluThrCysHisSerValGlyAlaAlaTyrLeu    465470475480    GluSerLeuProLeuGlnAspAlaSerProAlaGlyGlyProSerSer    485490495    ProArgAspLeuProGluProArgValSerThrGluHisThrAsnAsn    500505510    LysIleGluLysIleTyrIleMetLysAlaAspThrValIleValGly    515520525    ThrValLysAlaGluLeuProGluGlyArgGlyLeuAlaGlyProAla    530535540    GluProGluLeuGluGluGluLeuGluAlaAspHisThrProHisTyr    545550555560    ProGluGlnGluThrGluProProLeuGlySerCysSerAspValMet    565570575    LeuSerValGluGluGluGlyLysGluAspProLeuProThrAlaAla    580585590    SerGlyLys    595    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Homo sapiens    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TTCCCACAGGATCGACCC18    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Homo sapiens    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    CTCCCACGGGGAAG14    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 16 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Homo sapiens    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    CACCGGAGGGCCTGCA16    (2) INFORMATION FOR SEQ ID NO:6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 14 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Homo sapiens    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    CTGCCTCTGGAAAG14    (2) INFORMATION FOR SEQ ID NO:7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 122 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Protein    (B) LOCATION: 1..122    (D) OTHER INFORMATION: /note= "CD30 (1A), see Fig. 5"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    ThrCysHisGlyAsnProSerProTyrTyrAspLysAlaValArgArg    151015    CysCysTyrArgCysProMetGlyLeuPheProThrGlnGlnCysPro    202530    GlnArgProThrAspCysArgGlnCysGluProAspTyrTyrLeuAsp    354045    GluAlaAspArgCysThrAlaCysValThrCysSerArgAspAspLeu    505560    ValGluLysThrProCysAlaTrpAsnSerSerArgValCysGluCys    65707580    ArgProGlyMetPheCysSerThrSerAlaValAsnSerCysAlaArg    859095    CysPhePheHisSerValCysProAlaGlyMetIleValLysPhePro    100105110    GlyThrAlaGlnLysAsnThrValCysGlu    115120    (2) INFORMATION FOR SEQ ID NO:8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 120 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Protein    (B) LOCATION: 1..120    (D) OTHER INFORMATION: /note= "CD30 (1B), see Fig. 5"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    AlaSerLysLeuThrArgAlaProAspSerProSerSerValGlyArg    151015    ProSerSerAspProGlyLeuSerProThrGlnProCysProGluGly    202530    SerGlyAspCysArgGlnCysGluProAspTyrTyrLeuAspGluAla    354045    GlyArgCysThrAlaCysValSerCysSerArgAspAspLeuValGlu    505560    LysThrProCysAlaTrpAsnSerSerArgThrCysGluCysArgPro    65707580    GlyMetIleCysAlaThrSerAlaThrAsnSerCysAlaArgCysVal    859095    ProTyrProIleCysAlaAlaGluThrValThrLysProGlnAspMet    100105110    AlaGluLysAspThrThrPheGlu    115120    (2) INFORMATION FOR SEQ ID NO:9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 164 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Protein    (B) LOCATION: 1..164    (D) OTHER INFORMATION: /note= "TNFR2, see Fig. 5"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    ThrCysArgLeuArgLeuGluTyrTyrAspGlnThrAlaGlnMetCys    151015    CysSerLysCysSerProGlyGlnHisAlaLysValPheCysThrLys    202530    ThrSerAspThrValCysAspSerCysGluAspSerThrTyrThrGln    354045    LeuTrpAsnTrpValProGluCysLeuSerCysGlySerArgCysSer    505560    SerAspGlnValGluThrGlnAlaCysThrArgGluGlnAsnArgIle    65707580    CysThrCysArgProGlyTrpTyrCysAlaLeuSerLysGlnGluGly    859095    CysArgLeuCysAlaProLeuArgLysCysArgProGlyPheGlyVal    100105110    AlaArgProGlyThrGluThrSerAspValValCysLysProCysAla    115120125    ProGlyThrPheSerAsnThrThrSerSerThrAspIleCysArgPro    130135140    HisGlnIleCysAsnValValAlaIleProGlyAsnAlaSerMetAsp    145150155160    AlaValCysThr    (2) INFORMATION FOR SEQ ID NO:10:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 154 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Protein    (B) LOCATION: 1..154    (D) OTHER INFORMATION: /note= "TNFR1, see Fig. 5"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    ValCysProGlnGlyLysTyrIleHisProGlnAsnAsnSerIleCys    151015    CysThrLysCysHisLysGlyThrTyrLeuTyrAsnAspCysProGly    202530    ProGlyGlnAspThrAspCysAspGluCysGluSerGlnSerPheThr    354045    AlaSerGluAsnHisLeuArgHisCysLeuSerCysSerLysCysArg    505560    LysGluMetGlyGlnValGluIleSerSerCysThrValAspArgAsp    65707580    ThrValCysGlyCysArgLysAsnGlnTyrArgHisTyrTrpSerGlu    859095    AsnLeuPheGlnCysPheAsnCysSerLeuCysLeuAsnGlyThrVal    100105110    HisLeuSerGlyGlnGluLysGlnAsnThrValCysThrCysHisAla    115120125    GlyPhePheLeuArgGluAsnGluCysValSerCysGlyAsnCysLys    130135140    LysSerLeuGluCysThrLysLeuCysLeu    145150    (2) INFORMATION FOR SEQ ID NO:11:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 159 amino acids    (B) TYPE: amino acid    (C) STRANDEDNESS: Not Relevant    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: NO    (v) FRAGMENT TYPE: internal    (ix) FEATURE:    (A) NAME/KEY: Protein    (B) LOCATION: 1..159    (D) OTHER INFORMATION: /note= "NGFR, see Fig. 5"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    AlaCysProThrGlyLeuTyrThrHisSerGlyGluCysCysLysAla    151015    CysAsnLeuGlyGluGlyValAlaGlnProCysGlyAlaAsnGlnThr    202530    ValCysGluProCysLeuAspAsnValThrPheSerAspValValSer    354045    AlaThrGluProCysLysProCysThrGluCysLeuGlyLeuGlnSer    505560    MetSerAlaProCysValGluAlaAspAspAlaValCysArgCysAla    65707580    TyrGlyTyrTyrGlnAspGluThrThrGlyHisCysGluAlaCysSer    859095    ValCysGluValGlySerGlyLeuValPheSerCysGlnAspLysGln    100105110    AsnThrValCysGluGluCysProAspGlyThrTyrSerAspGluAla    115120125    AsnHisValAspProCysLeuProCysThrValCysGluAspThrGlu    130135140    ArgGlnLeuArgGluCysThrArgTrpAlaAspAlaGluCysGlu    145150155    __________________________________________________________________________

We claim:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a sequence encoding the amino acid sequence ofSEQ. ID. NO.:
 2. 2. A recombinant vector comprising an isolated nucleicacid of claim
 1. 3. A cell transformed with a recombinant vector ofclaim
 2. 4. An isolated nucleic acid molecule comprising apolynucleotide having the nucleotide sequence of SEQ. ID. NO.
 1. 5. Arecombinant vector comprising an isolated nucleic acid of claim
 4. 6. Acell transformed with a recombinant vector of claim
 5. 7. A method forproducing a CD30 protein comprising:i) culturing a transformed cell ofclaim 3 or 6; and ii) recovering CD30 protein produced by the culturedhost cell.
 8. A method for preparing a hybridization probe comprising:i)isolating a restriction fragment of the DNA molecule of claim 1 or claim4 and its complementary strand; ii) nick-translating the restrictionfragment of step (i) in the presence of a labeled nucleotide to obtain alabeled hybridization probe.
 9. A hybridization probe prepared accordingto the method of claim
 8. 10. An isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence selected fromthe group consisting ofa sequence encoding the portion of the amino acidsequence of SEQ. ID. NO. 2 from residue 19 to residue 379; a sequenceencoding the portion of the amino acid sequence of SEQ. ID. NO. 2 fromresidue 408 to residue 595; a sequence encoding the portion of the aminoacid sequence of SEQ. ID. NO. 2 from residue 19 to residue 93; asequence encoding the portion of the amino acid sequence of SEQ. ID. NO.2 from residue 112 to residue 416; the sequence from nucleotide 218 tonucleotide 1732 of SEQ. ID. NO. 1; the sequence from nucleotide 277 tonucleotide 1359 of SEQ. ID. NO. 1; and the sequence from nucleotide 1444to nucleotide 2007 of SEQ. ID. NO.
 1. 11. A recombinant vectorcomprising an isolated nucleic acid of claim
 10. 12. A cell transformedwith a recombinant vector of claim
 11. 13. A hybridization probecomprising the nucleic acid of claim 1 or 10 and a label selected fromthe group consisting of radioactive atoms, stain groups, or epitopesintroduced for systems based on light or enzymatic stain reactions fordetection of nucleic acids.
 14. An isolated nucleic acid moleculecomprising a polynucleotide having a nucleotide sequence selected fromthe group consisting ofportion I, which encodes a part of the amino acidsequence of SEQ. ID. NO. 2 between residue 19 and residue 93 thatpresents an epitope that is specifically bound by the Ki-1 monoclonalantibody; portion II, which encodes a part of the amino acid sequence ofSEQ. ID. NO. 2 between residue 112 and residue 416 that presents anepitope that is specifically bound by the Ber-H2 monoclonal antibody;and portion III, which encodes a part of the amino acid sequence of SEQ.ID. NO. 2 between residue 19 and residue 416 and that presents anepitope that is specifically bound by the Ki-1 monoclonal antibody andan epitope that is specifically bound by the Ber-H2 monoclonal antibody.15. A recombinant vector comprising an isolated nucleic acid of claim14.
 16. A cell transformed with a recombinant vector of claim
 15. 17. Anisolated nucleic acid encoding a fusion protein comprising a firstpolypeptide comprising an amino acid sequence selected from the groupconsisting ofthe portion of the amino acid sequence of SEQ. ID. NO. 2from residue 19 to residue 379; the portion of the amino acid sequenceof SEQ. ID. NO. 2 from residue 408 to residue 595; the amino acidsequence of SEQ. ID. NO. 2; the portion of SEQ. ID. NO. 2 betweenresidue 19 and residue 263 that presents an epitope that is specificallybound by the Ki-1 monoclonal antibody; that portion of SEQ. ID. NO. 2between residue 112 and residue 416 that presents an epitope that isspecifically bound by the Ber-H2 monoclonal antibody; and that portionof SEQ. ID. NO. 2 between residue 19 and residue 416 and that presentsan epitope that is specifically bound by the Ki-1 monoclonal antibodyand an epitope that is specifically bound by the Ber-H2 monoclonalantibody;and a second polypeptide.
 18. An isolated nucleic acid of claim17, wherein said second polypeptide is a bacterial protein.
 19. Arecombinant vector comprising an isolated nucleic acid of claim
 18. 20.A cell transformed with a recombinant vector of claim
 19. 21. Anisolated nucleic acid of claim 17, wherein said second polypeptide isβ-galactosidase.
 22. A recombinant vector comprising an isolated nucleicacid of claim
 21. 23. A cell transformed with a recombinant vector ofclaim
 22. 24. A recombinant vector comprising an isolated nucleic acidof claim
 17. 25. A cell transformed with a recombinant vector of claim24.
 26. An isolated nucleic acid encoding a fusion protein comprising(1) the extracellular portion of human CD30 consisting essentially ofresidues 19 to 379 of SEQ. ID. NO. 2 and (2) a second polypeptide.
 27. Arecombinant vector comprising an isolated nucleic acid of claim
 26. 28.A cell transformed with a recombinant vector of claim
 27. 29. Arecombinant vector according to any one of claims 2, 5, 11, 15, 19, 22,24 and 27 which further comprises an inducible promoter.
 30. Atransformed cell of any one of claims 3, 6, 12, 16, 20, 23, 25 and 28that is a eukaryotic cell.
 31. A transformed cell of claim 30 that is amammalian cell.
 32. A method for producing a CD30 protein, a portion ofa CD30 protein, or a fusion protein comprising a CD30 protein orcomprising a portion of CD30 protein, comprising:i) culturing atransformed cell of any one of claims 12, 16, 20, 23, 25 and 28; and ii)recovering CD30 protein produced by the cultured host cell.